The Inhibitory Effect of Blue Light on Phagocytic Activity by ARPE-19 Cells.

Chronic exposure of the retina to short wavelength visible light is a risk factor in pathogenesis of age-related macular degeneration. The proper functioning and survival of photoreceptors depends on efficient phagocytosis of photoreceptor outer segments (POS) by retinal pigment epithelium. The purpose of this study was to analyze the phagocytic activity of blue light-treated ARPE-19 cells, and to examine whether the observed effects could be related to altered levels of POS phagocytosis receptor proteins and/or to oxidation of cellular proteins and lipids. POS phagocytosis was measured by flow cytometry. Phagocytosis receptor proteins αv and ß5 integrin subunits and Mer tyrosine kinase (MerTK) were quantified by western blotting. The intact functional heterodimer αvß5 was quantified by immunoprecipitation followed by immunoblotting. Cellular protein and lipid hydroperoxides were analyzed by coumarin boronic acid probe and iodometric assay, respectively. Cell irradiation induced reversible inhibition of specific phagocytosis and transient reductions in phagocytosis receptor proteins. Full recovery of functional heterodimer was apparent. Significant photooxidation of cellular proteins and lipids was observed. The results indicate that transient inhibition of specific phagocytosis by blue light could be related to the reduction in phagocytosis receptor proteins. Such changes may arise from oxidative modifications of cell phagocytic machinery components.

Melanopsin Signaling Pathway in HEK293 Cell Line with Stable Expression of Human Melanopsin: Possible Participation of Phospholipase C beta 4 and Diacylglycerol.

Melanopsin, a member of the G protein-coupled receptors family, is involved in non-image-forming functions including circadian rhythm, sleep regulation and pupil response. In spite of significant research efforts, the signaling cascade involving melanopsin photoactivation remains poorly characterized. Here, we analyzed the effects of photoactivation of melanopsin on phospholipase C (PLC) and diacylglycerol. As an in vitro model, HEK293 cells with stable expression of human melanopsin were used. Although both the PLCß1 and PLCß4 subtypes were activated by the cell exposure to blue light, only PLCß4 appeared to play a significant role in the studied melanopsin signaling pathway. We have demonstrated, for the first time, that cells expressing human melanopsin and enriched with 11-cis-retinal exhibited significantly increased diacylglycerol level. To determine the role of phospholipase C and involvement of diacylglycerols, two approaches were employed: inhibition of the G protein and phospholipase C (using the BIM-46187 and U73122 inhibitors, respectively), and gene silencing using siRNA of PLCß1 and PLCß4 . While silencing the PLCß4 gene and using U73122 inhibited the diacylglycerol and calcium ion responses, the FOS gene expression level was only partially reduced. These results may facilitate a better understanding of the role of phospholipase C and diacylglycerols in the melanopsin signaling pathway.

The effect of aging and antioxidants on photoreactivity and phototoxicity of human melanosomes: An in vitro study.

Aging may significantly modify antioxidant and photoprotective properties of melanin in retinal pigment epithelium (RPE). Here, photoreactivity of melanosomes (MS), isolated from younger and older human donors with and without added zeaxanthin and α-tocopherol, was analyzed by electron paramagnetic resonance oximetry, time-resolved singlet oxygen phosphorescence, and protein oxidation assay. The phototoxic potential of ingested melanosomes was examined in ARPE-19 cells exposed to blue light. Phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was determined by flow cytometry. Irradiation of cells fed MS induced significant inhibition of the specific phagocytosis with the effect being stronger for melanosomes from older than from younger human cohorts, and enrichment of the melanosomes with antioxidants reduced the inhibitory effect. Cellular protein photooxidation was more pronounced in samples containing older melanosomes, and it was diminished by antioxidants. This study suggests that blue light irradiated RPE melanosomes could induce substantial inhibition of the key function of the cells-their specific phagocytosis. The data indicate that while photoreactivity of MS and their phototoxic potential increase with age, they could be reduced by selected natural antioxidants.

The Effect of Antioxidants on Photoreactivity and Phototoxic Potential of RPE Melanolipofuscin Granules from Human Donors of Different Age.

One of the most prominent age-related changes of retinal pigment epithelium (RPE) is the accumulation of melanolipofuscin granules, which could contribute to oxidative stress in the retina. The purpose of this study was to determine the ability of melanolipofuscin granules from younger and older donors to photogenerate reactive oxygen species, and to examine if natural antioxidants could modify the phototoxic potential of this age pigment. Electron paramagnetic resonance (EPR) oximetry, EPR-spin trapping, and time-resolved detection of near-infrared phosphorescence were employed for measuring photogeneration of superoxide anion and singlet oxygen by melanolipofuscin isolated from younger and older human donors. Phototoxicity mediated by internalized melanolipofuscin granules with and without supplementation with zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells by determining cell survival, oxidation of cellular proteins, organization of the cell cytoskeleton, and the cell specific phagocytic activity. Supplementation with antioxidants reduced aerobic photoreactivity and phototoxicity of melanolipofuscin granules. The effect was particularly noticeable for melanolipofuscin mediated inhibition of the cell phagocytic activity. Antioxidants decreased the extent of melanolipofuscin-dependent oxidation of cellular proteins and disruption of the cell cytoskeleton. Although melanolipofuscin might be involved in chronic phototoxicity of the aging RPE, natural antioxidants could partially ameliorate these harmful effects.

Photoreactivity of Bis-retinoid A2E Complexed with a Model Protein in Selected Model Systems.

The bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E) is formed as a byproduct of visual cycle in retinal pigment epithelium (RPE). It contributes to golden-yellow fluorescence of the age pigment lipofuscin, which accumulates in RPE. Lipofuscin can generate a variety of reactive oxygen species (ROS) upon blue-light excitation. Although in model systems photoreactivity of A2E has been determined to be low, this bis-retinoid exhibited significant phototoxicity in RPE cells in vitro. Although the mechanism of A2E-mediated phototoxicity remains mostly unknown, we hypothesize that formation of A2E-adducts with different biomolecules may play an important role. In this study, we investigated the photochemical reactivity of A2E and its complex with bovine serum albumin (BSA) using UV-Vis absorption and emission spectroscopy, EPR-spin trapping, EPR-oximetry, time-resolved singlet oxygen phosphorescence, and the fluorogenic CBA probe. Our data show that A2E after complexation with this model protein photogenerated an increased level of ROS, particularly singlet oxygen. We also demonstrated the ability of A2E to oxidize BSA upon excitation with blue light in aqueous model systems. The data suggest that pyridinium bis-retinoid could oxidatively modify cellular proteins under physiological conditions.

Quercetin protects ARPE-19 cells against photic stress mediated by the products of rhodopsin photobleaching.

Although the primary biological function of retina photoreceptors is to absorb light and provide visual information, exposure to intense light could increase the risk of phototoxic reactions mediated by rhodopsin photobleaching products (RPBP) that might accumulate in photoreceptor outer segments (POS). Here we investigated whether quercetin can modify the phototoxic potential of RPBP under in vitro photic stress conditions. ARPE-19 cells or quercetin enriched cultures pre-loaded with rhodopsin-rich POS isolated from bovine retinas were irradiated with green light to photobleach rhodopsin, and subsequently with blue light. Survival of cells was determined by MTT assay and propidium iodide staining. Changes in mitochondrial membrane potential (MMP) were assessed by JC-1 staining. Protein hydroperoxides, formed by photosensitized oxidation, mediated by RPBP, were analyzed in cells and in a model system with bovine serum albumin (BSA), using the coumarin boronic acid fluorogenic probe. The effect of photic stress on specific phagocytosis of RPE cells was determined by flow cytometry. Photoreactivity of POS with and without quercetin was analyzed by EPR oximetry and EPR spin trapping. Cytotoxicity measurements and MMP analyses confirmed that supplementation with quercetin protected ARPE-19 cells against photic stress mediated by rhodopsin-rich POS. Quercetin significantly reduced the inhibitory effect of RPBP-mediated stress on POS phagocytosis and the RPBP ability to photooxidize cellular proteins or BSA. The data support the hypothesis that quercetin may efficiently diminish the phototoxic action of retinoids, necessary for restoring the phagocytic function of ARPE-19 cells.

Lipofuscin-mediated photic stress inhibits phagocytic activity of ARPE-19 cells; effect of donors' age and antioxidants.

The risk of chronic oxidative stress in the retinal pigment epithelium (RPE) increases with age due to accumulation of the photoreactive age pigment lipofuscin (LFG). Here, we asked whether sublethal and weakly lethal photic stress, induced by irradiation of ARPE-19 cells containing phagocytised LFG, affected the cell specific phagocytic activity, which is critically important for proper functioning and survival of the retina, and if natural antioxidants could modify the observed outcomes. ARPE-19 cells preloaded with LFG isolated from human donors of different age or containing LFG enriched with zeaxanthin and α-tocopherol (LFG-A), were irradiated with blue light. Phagocytosis of fluorescein-5-isothiocyanate (FITC)-labelled photoreceptor outer segments was determined by flow cytometry. Photoreactivity of LFG and LFG-A was analysed by measuring photoconsumption of oxygen and photogeneration of singlet oxygen mediated by the granules. LFG-mediated photic stress in ARPE-19 cells induced significant inhibition of their specific phagocytosis. The inhibitory effect increased with age of LFG donors and was reduced by enrichment of the granules with antioxidants. Oxygen consumption and generation of singlet oxygen induced by the photoexcited LFG increased with donor's age and was partially quenched by antioxidants. Although the phototoxic potential of lipofuscin increased with age, natural antioxidants reduced photoreactivity of LFG and their efficiency to induce oxidative stress. This study has demonstrated, for the first time, that mild oxidative stress, mediated by the age pigment lipofuscin, impairs specific phagocytic activity of RPE, and that natural antioxidants can protect this important cellular function by reducing lipofuscin photoreactivity.

Zeaxanthin and α-tocopherol reduce the inhibitory effects of photodynamic stress on phagocytosis by ARPE-19 cells.

Zeaxanthin and α-tocopherol have been previously shown to efficiently protect liposomal membrane lipids against photosensitized peroxidation, and to protect cultured RPE cells against photodynamic killing. Here the protective action of combined zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells subjected to photodynamic (PD) stress mediated by rose Bengal (RB) or merocyanine-540 (MC-540) at sub-lethal levels. Stress-induced cytotoxicity was analyzed by the MTT assay. The peroxidation of membrane lipids was determined by HPLC-EC (Hg) measurements of cholesterol hydroperoxides using cholesterol as a mechanistic reporter molecule. The specific phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was measured by flow cytometry, and the levels of phagocytosis receptor proteins αv integrin subunit, ß5 integrin subunit and MerTK were quantified by Western blot analysis. Cytotoxicity measures confirmed that PD stress levels used for phagocytosis analysis were sub-lethal and that antioxidant supplementation protected against higher, lethal PD doses. Sub-lethal PD stress mediated by both photosensitizers induced the accumulation of 5α-OOH and 7α/ß-OOH cholesterol hydroperoxides and the addition of the antioxidants substantially inhibited their accumulation. Antioxidant delivery prior to PD stress also reduced the inhibitory effect of stress on POS phagocytosis and partially reduced the stress-induced diminution of phagocytosis receptor proteins. The use of a novel model system where oxidative stress was induced at sub-lethal levels enable observations that would not be detectable using lethal stress models. Moreover, novel observations about the protective effects of zeaxanthin and α-tocopherol on photodynamic damage to ARPE-19 cell membranes and against reductions in the abundance of receptor proteins involved in POS phagocytosis, a process essential for photoreceptor survival, supports the importance of the antioxidants in protecting of the retina against photooxidative injury.

Photic injury to cultured RPE varies among individual cells in proportion to their endogenous lipofuscin content as modulated by their melanosome content.

PURPOSE: We determined whether photic stress differentially impairs organelle motility of RPE lipofuscin and melanin granules, whether lethal photic stress kills cells in proportion to lipofuscin abundance, and whether killing is modulated by melanosome content. METHODS: Motility of endogenous lipofuscin and melanosome granules within the same human RPE cells in primary culture was quantified by real-time imaging during sublethal blue light irradiation. Cell death during lethal irradiation was quantified by dynamic imaging of the onset of nuclear propidium iodide fluorescence. Analyzed were individual cells containing different amounts of autofluorescent lipofuscin, or similar amounts of lipofuscin and a varying content of phagocytized porcine melanosomes, or phagocytized black latex beads (control for light absorbance). RESULTS: Lipofuscin granules and melanosomes showed motility slowing with mild irradiation, but slowing was greater for lipofuscin. On lethal irradiation, cell death was earlier in cells with higher lipofuscin content, but delayed by the copresence of melanosomes. Delayed death did not occur with black beads, suggesting that melanosome protection was due to properties of the biological granule, not simple screening. CONCLUSIONS: Greater organelle motility slowing of the more photoreactive lipofuscin granule compared to melanosomes suggests that lipofuscin mediates mild photic injury within RPE cells. With lethal light stress endogenous lipofuscin mediates killing, but the effect is cell autonomous and modulated by coincident melanosome content. Developing methods to quantify the frequency of individual cells with combined high lipofuscin and low melanosome content may have value for predicting the photic stress susceptibility of the RPE monolayer in situ.

Photosensitized oxidative stress to ARPE-19 cells decreases protein receptors that mediate photoreceptor outer segment phagocytosis.

PURPOSE: To determine whether previously shown photodynamic (PD)-induced inhibition of specific photoreceptor outer segment (POS) phagocytosis by ARPE-19 cells is associated with reductions in receptor proteins mediating POS phagocytosis, and if PD treatment with merocyanine-540 (MC-540) produces additional effects leading to its inhibition of nonspecific phagocytosis. METHODS: ARPE-19 cells preloaded with MC-540 or rose bengal (RB) were sublethally irradiated with green light. Phagocytosis of POS was measured by flow cytometry and POS receptor proteins (Mer tyrosine kinase receptor [MerTK] and integrin subunits αv and ß5) and ß-actin were quantified by Western blotting at 0.5 and 24 hours after irradiation, with comparison to samples from nonsensitized control cultures. The intact integrin heterodimer αvß5 was quantified by immunoprecipitation followed by blotting. The distribution of N-cadherin, ZO-1, and F-actin was visualized by fluorescence microscopy. RESULTS: Mild PD stress mediated by both photosensitizers that elicits no significant morphologic changes produces transient and recoverable reductions in MerTK. The individual αv and ß5 integrin subunits are also reduced but only partially recover. However, there is sufficient recovery to support full recovery of the functional heterodimer. Light stress mediated by MC-540 also reduced levels of actin, which is known to participate in the internalization of particles regardless of type. CONCLUSIONS: After PD treatment POS receptor protein abundance and phagocytosis show a coincident in time reduction then recovery suggesting that diminution in receptor proteins contributes to the phagocytic defect. The additional inhibition of nonspecific phagocytosis by MC-540-mediated stress may result from more widespread effects on cytosolic proteins. The data imply that phagocytosis receptors in RPE cells are sensitive to oxidative modification, raising the possibility that chronic oxidative stress in situ may reduce the efficiency of the RPE's role in photoreceptor turnover, thereby contributing to retinal degenerations.

Photobleaching of retinal pigment epithelium melanosomes reduces their ability to inhibit iron-induced peroxidation of lipids.

Melanin in the human retinal pigment epithelium (RPE) is believed to play an important photoprotective role. However, unlike in skin, melanosomes in the RPE are rather long-lived organelles, which increases their risk of modifications resulting from significant fluxes of light and high oxygen tension. In this work, we subjected purified bovine RPE melanosomes to prolonged aerobic exposure with intense visible and near ultraviolet radiation and studied the effects of irradiation on the melanosome's capacity to inhibit peroxidation of lipids induced by iron/ascorbate. We found that control, untreated melanosomes show a concentration-dependent inhibition of the accumulation of lipid hydroperoxides and the accompanying consumption of oxygen, but photolysed melanosomes lose their antioxidant efficiency and even became prooxidant. The prooxidant action of partially photobleached melanosomes was observed for pigment granules with a melanin content reduced by about 50% compared with untreated melanosomes, as determined by electron spin resonance spectroscopy. We have previously shown that a similar loss in the content of the RPE melanin occurs during human lifetime, which may suggest that the normal antioxidant properties of human RPE melanin become compromised with aging.